Blood cancers such as leukemia, Hodgkin and non-Hodgkin lymphoma, and myeloma will account for nearly 10% of the 1.5 million new cancer cases diagnosed each year in the United States. Currently the most effective treatment against these diseases is hematopoietic stem cell (HSC) transplantation. However, the proper harvest of these cells remains inefficient and compatibility issues make it difficult to find an appropriate match for HSC transplants. With proper control of their differentiation, embryonic stem cells (ESCs) may provide a powerful cell source for HSCs and other cell types needed for tissue regeneration. Although extensive studies have been done to elucidate the roles of various soluble factors in the differentiation of HSCs and ESCs, the role of the extracellular matrix (ECM) niche, especially the biophysical aspects, has not been well understood. The purpose of this study is to develop a method of regulating ESC differentiation through the manipulation of matrix stiffness. Previous studies have demonstrated that stiffness of the extracellular matrix (ECM) guides adult mesenchymal stem cell (MSC) differentiation into osteoblastic, skeletal muscular and neural lineages, suggesting that the mechanical properties of the ECM, in addition to chemical factors, are critical for stem cell differentiation. However, whether ECM stiffness regulates ESC differentiation into other cell types and the underlying molecular mechanisms require further investigation. We propose to use a tunable polyacrylamide system with murine ESCs to study the regulation of ESC differentiation, specifically into the hematopoietic lineage. Our pilot studies have demonstrated that softer matrix surfaces allow the ESCs to differentiate more efficiently into cells with hematopoietic potential. From this data we hypothesize that the mechanical properties of the ECM can regulate stem cell differentiation into the hematopoietic lineage. Furthermore, we hypothesize that the type of biochemical interaction between the ESCs and the matrix components affects the efficacy with which these ESC-derived hematopoietic cells are generated. We propose two specific aims to prove our hypotheses (Figure 1): 1. Determine how mechanical and chemical properties of the ECM regulate ESC differentiation into fetal liver kinase-1 (Flk-1) positive hemangioblasts. 2. Investigate how mechanical and chemical properties of the ECM affect differentiation of Flk-1+ hemangioblasts into hematopoietic cells. To better elucidate the mechanisms, mouse ESCs will be used in the proposed studies. The hematopoietic differentiation pathways in the murine system have been established, and a specific ESC line will allow simultaneous monitoring of mesoderm and hematopoietic differentiation. PUBLIC HEALTH RELEVANCE: Success of the project could shed light on the roles of the ECM niche (both mechanical and chemical properties) on ESC differentiation into hematopoietic cells, and provide a platform for the development of stem cell therapies and tissue regeneration, which in turn could lead to cures to many diseases, including blood cancers.